Experiments are proposed to characterize gene regulation in the methionine biosynthetic pathway in E. coli K12. A system for cell-free DNA-dependent synthesis of one of the enzymes of the pathway, the non-B12 transmethylase (metE) will be established. Attempts to establish in vitro regulation of expression of this gene will be made by addition of cell extracts containing the methionine repressor (metJ) in combination with methionine and other possible effectors. Once in vitro regulation is established purification of the methionine repressor can be done. In order to enrich for methionine repressor as an aid to purification, the metJ gene will be cloned onto a multicopy plasmid. A second approach toward understanding metE regulation will be to characterize metE DNA by nucleotide sequence analysis. Therefore, using recombinant RNA methodology the shortest possible metE-carrying DNA fragment will be cloned onto an appropriate plasmid. Since such a fragment must be at least 2.6 kilobases (as calculated from the amino acid content of the enzyme monomer) further restriction analysis will be done in order to obtain fragments which are short enough to sequence.